MGC Frequently Asked Questions

Usually between 8:00am and 5:00pm, email DMPI-MGC@dm.duke.edu to schedule.

Please see the MGC Project Guidelines

Please see the MGC Service Request Guide below:

• Single cell/nuclei projects run for 2-5 days, depending on the assay, then they are sent to the Sequencing Core, whose turnaround time varies
• Visium projects are processed for 3 days, depending on the assay, then they are sent to the Sequencing Core, whose turnaround time varies
• Estimated turnaround time for single cell/nuclei & Visium basic analysis is 2 weeks, once we receive data back from the Sequencing Core
• Xenium projects are processed for 3 days and then imaged for an additional 3-6 days then customers will have data transferable to them
• SNP genotyping projects run for 2 days, then raw data results are shared with customer
• Quanterix Simoa® Digital Immunoassay Biomarker Detection projects run for 1-2 days, depending on project size, then raw data results are shared with the customer

Yes, some instruments are. Prior to gaining walk-up access, you will require training with an MGC staff member. To organize this training, please email us at DMPI-MGC@dm.duke.edu

This will be dependent on the desired downstream assay and sample type. In general:

• Fresh Frozen: Collect tissue, rinse, in some cases perfuse, with ice cold PBS then flash freeze in isopentane bath. Storage in liquid nitrogen is preferred, but -80 is also acceptable. For downstream chromatin-based applications, please store in liquid nitrogen. 
  • 10x genomics guide here:  https://kb.10xgenomics.com/hc/en-us/articles/360051025792-How-do-you-isolate-nuclei-from-snap-frozen-tissue-for-3-gene-expression-profiling
• Formalin Fixed: Collect tissue and immediately fix in 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA). Time in fixative is dependent on tissue type. Once tissue is fixed, dehydrate and embed in paraffin wax.
  • See guide here: https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/ 

• The MGC will store generated cDNA and libraries for 3 months, after which they will be destroyed. Leftover cells for scRNAseq will not be saved.
• Please contact MGC if you’d like your sample and/or libraries returned to you.

The MGC will store data for 3 months, after which it will be discarded.

“We would like to acknowledge the assistance of the Duke Molecular Physiology Institute Molecular Genomics Core for the generation of data for the manuscript.”

• We are located in the Duke Molecular Physiology Institute (Carmichael Building) at 300 N Duke Street in Bay 51.
• When you’re driving on N Duke Street, pass the building and take your first right onto W Corporation Street, then your first right will bring you to our loading dock. Above the loading dock, there is a ‘52’ above the door. Once you’ve arrived, please contact your designated MGC staff member for sample drop-off.

Archived FFPE samples are compatible with the single cell Flex assay. Archival FFPE slides are compatible with 10x Genomics Visium CytAssist v1 and HD assays as well as Xenium. RNA QC prior to running a gene expression assay is highly recommended.

Please request methods by emailing us at DMPI-MGC@dm.duke.edu.

DV200 is the percentage of fragments in an RNA sample >200bp. This metric can be produced from an Agilent Bioanalyzer or Tapestation using the RNA kits. This metric is used to assess fixed samples because the process of fixation causes the samples to be fragmented and an RNA integrity number would be consistently low for these samples. Assays such as Xenium or Visium HD employ probe-based chemistry to detect transcripts instead of sequencing captured poly-adenylated mRNA so intact RNA molecules are not as necessary for the assay to perform well.

We suggest > 80% viability for cells and >80% intactness for nuclei, assessed via AOPI and fluorescent cell counter/microscope.

Our queue is usually 4-6 weeks, but it is best to schedule a run as soon as you know an approximate day you will be ready. Send us an email if you have any unusual circumstances so we can work with you to decide how to proceed.

LINK TO PDF PROJECT GUIDELINES

Email the MGC (DMPI-MGC@dm.duke.edu) to set up an initial consult

With the current 10x Genomics GEM-X chemistry, you can target 500-20k cells/nuclei for capture. Typically, we request at least 3x the targeted number of cells/nuclei so that we have enough for counting in addition to the assay.

Certain circumstances that involve additional washing steps require many more cells be provided, such as the Flex assay.

Yes, we would require your SOPs and additional advanced notice to plan for use of a biosafety hood.

FFPE sections for flex are good for 1 year at 4°C and FF sections for nuclei are stable at 80°C or liquid nitrogen. 

We do have the capability to prepare slides for spatial transcriptomics, however we do not have a histologist/pathologist on staff to perform morphological/pathological assessments of specimens. If your project requires tissue to be assessed for morphological or pathological features, please reach out to the BRPC for assistance for tissue services (brpc@win.duke.edu). If your sample blocks are already faced and at the location to be collected, the MGC can perform your cutting services.

As many that can fit without overlapping each other or the fiducial boundaries in a 10mm x 22mm area.

Two Xenium slides per run.

https://www.10xgenomics.com/support/software/xenium-panel-designer/latest/tutorials/pre-designed-xenium-v1-parts

https://www.10xgenomics.com/support/software/xenium-panel-designer/latest/tutorials/pre-designed-xenium-prime-5k-parts

The Xenium Prime workflow is designed for higher-plexity probe capabilities (5000 genes) but is limited to Mouse or Human panels. Xenium v1 is limited to 480 genes currently but is more flexible with species.

Fresh frozen in OCT and Formalin-fixed paraffin embedded tissues. Fixed frozen in OCT is possible, but not fully supported by 10x Genomics.

5um for FFPE, 10um for Frozen

Yes, there is no concern for cross contamination by placing sections from unique individuals onto the same Xenium slide.

Yes but due to the limit in how many individual samples can be uniquely imaged in their own field of view, depending on how many samples are in the TMA, you may be required to group samples by treatment or condition. The limit is 8 imaging fields per slide so if you had a TMA of 24 samples, we would require you to segregate them into up to 8 groups ie: Group 1 Controls, Group 2 Treatment A, Group 3 Treatment B and so on.

Visium runs are in batches of four samples. In instances where you need to run more than four samples, multiple run dates are scheduled.

Yes you can run 2 samples but either you would still purchase an entire 4 reaction kit or you would wait for us to find someone else with a project of 2 samples to run with yours. This is not a common practice as wait times to find a run partner can be long and few people request to run only 2 samples.

Fresh frozen in OCT, Formalin-fixed paraffin embedded tissues and Fixed frozen in OCT.

5um for FFPE, 10-20um for Frozens

The main difference between the two assays is the resolution. The v1 assay has a lawn of 55um oligo-laden spots with 100um distance between spots center to center while the HD assay has 2um squares abutted next to each other so there is no dead space in between.

Yes, you can design custom probes according to 10x Genomics guidance and send these to us with your samples to spike into the assay during processing. However, we cannot guarantee success of probe spikes because they are not produced and validated by 10x Genomics.

See link to 10x Genomics custom probe design help below:

https://www.10xgenomics.com/support/flex-gene-expression/documentation/steps/experimental-design-and-planning/custom-probe-design-for-visium-spatial-gene-expression-and-chromium-single-cell-gene-expression-flex